A new platform for high-throughput therapy testing on iPSC-derived lung progenitor cells from cystic fibrosis patients

For those people with cystic fibrosis carrying rare CFTR mutations not responding to currently available therapies, there is an unmet need for relevant tissue models for therapy development. Here, we describe a new testing platform that employs patient-specific induced pluripotent stem cells (iPSCs) differentiated to lung progenitor cells that can be studied using a dynamic, high-throughput fluorescence-based assay of CFTR channel activity. Our proof-of-concept studies support the potential use of this platform, together with a Canadian bioresource that contains iPSC lines and matched nasal cultures from people with rare mutations, to advance patient-oriented therapy development. Interventions identified in the high-throughput, stem cell-based model and validated in primary nasal cultures from the same person have the potential to be advanced as therapies

Stimulation of hair growth using conditioned media derived from dental pulp stem cells expanded in serum free media / Tarini Nawamalie Abeysinghe Gunawardena

Introduction: Alopecia is a clinical condition caused by the hair loss and may result in baldness. The current treatment methods for this condition involve the use of drugs or natural substances. However, hair loss may be accelerated upon the discontinuation of medication. Alternatively, alopecia surgery could also be carried out though it has its limitations in the number of surgeries and the number of hair strands transplanted. Stem cell therapy, a recently emerged approach for alopecia treatment involves tedious procedures, complicated safety and quality management, low survival rate of transplanted cells and potential adverse immunological responses for the recipients. As an alternative, use of paracrine factors secreted by the stem cells showed promise in therapeutic applications. Paracrine factors involved in regulation of hair growth have been observed to be secreted by stem cells, in the culture media, termed as conditioned media (CM). Objectives: In this study we first verified the mesenchymal stem cell (MSC) like properties of human exfoliated or extracted deciduous dental pulp (SHED) and hair follicle stem cells (HFSCs) cultured in different concentrations of serum supplemented in DMEM-KO and in chemically defined media namely, STK2 (TwoCELLs, Japan). Secondly, hair growth regulatory paracrine factor profiles of CM prepared through culture of SHED and HFSCs in the respective media were ascertained. Lastly, the potential of SHED- and HFSC-CM to stimulate hair growth under in vitro and in vivo conditions were evaluated. Methods: SHED (n=3) and HFSCs (n=3) were cultured in, STK2 and other media combinations; DMEM-KO+10% FBS, DMEM-KO+10% FBS+bFGF, STK2+2% FBS and profiled for the presence of positive hair regulatory factors namely SDF-1, VEGF, HGF, PDGF-B and negative hair regulatory factors namely TGF-β, bFGF, TNF-, IL-1 and BDNF. The potential of the prepared CM to stimulate hair growth was evaluated based on the paracrine profile and the observed in vitro hair growth dynamics. The administration of the CM media via subcutaneous injection to telogen-staged synchronized 7-week old C3H/HeN female mice was carried out to further study the potential of the CM to stimulate hair growth in vivo. Results: SHED and HFSCs maintained their MSC characteristics in all media combinations. Cells cultured in STK2 based media showed better population doubling time, higher viability and better maintenance of MSC characteristics in comparison to cells cultured in DMEM-KO based media. SHED and HFSCs showed higher growth kinetics at passages 3 and 4. Cells cultured in DMEM-KO based media showed a higher expression of positive hair regulatory factors; SDF-1, VEGF, HGF, PDGF-B than cells cultured in STK2 based media. However, the difference was not statistically significant. Moreover, STK2 based CM contained only two negative hair regulatory factors, TNF-, IL-1 while DMEM-KO based CM media contained all negative hair regulatory factors that were tested. The in vitro studies confirmed that treatment with CM of passage 3 cells prepared in STK2 based media resulted in a significantly higher number of anagen-staged hair follicles (p< 0.05) and a significantly lower number of telogen-staged hair follicles (p< 0.05). Administration of SHED-CM prepared in STK2 to C3H/HeN mice resulted in a significantly faster stimulation of hair growth in comparison to the HFSC-CM prepared in STK2 (p< 0.05). Conclusions: Within the limitations of the study, STK2 is a better media for the expansion of both SHED and HFSCs and the maintenance of stem cell characteristics in comparison to DMEM-KO based media. SHED-CM prepared using STK2, significantly enhanced the stimulation of hair growth compared to HFSC-CM

Dental derived stem cell conditioned media for hair growth stimulation

Alopecia is a clinical condition caused by excessive hair loss which may result in baldness, the causes of which still remain elusive. Conditioned media (CM) from stem cells shows promise in regenerative medicine. Our aim was to evaluate the potential CM of dental pulp stem cells obtained from human deciduous teeth (SHED-CM) to stimulate hair growth under in vitro and in vivo conditions. SHED and hair follicle stem cells (HFSCs) (n = 3) were cultured in media combinations; i) STK2, ii) DMEM-KO+10% FBS, iii) STK2+2% FBS and profiled for the presence of positive hair growth-regulatory paracrine factors; SDF-1, HGF, VEGF-A, PDGF-BB and negative hair growth-regulatory paracrine factors; IL-1α, IL-1β, TGF-β, bFGF, TNF-α, and BDNF. The potential of CM from both cell sources to stimulate hair growth was evaluated based on the paracrine profile and measured dynamics of hair growth under in vitro conditions. The administration of CM media to telogen-staged synchronized 7-week old C3H/HeN female mice was carried out to study the potential of the CM to stimulate hair growth in vivo. SHED and HFSCs cultured in STK2 based media showed a shorter population doubling time, higher viability and better maintenance of MSC characteristics in comparison to cells cultured in DMEM-KO media. STK2 based CM contained only two negative hair growth-regulatory factors; TNF-α, IL-1 while DMEM-KO CM contained all negative hair growth-regulatory factors. The in vitro study confirmed that treatment with STK2 based media CM from passage 3 SHED and HFSCs resulted in a significantly higher number of anagen-staged hair follicles (p<0.05) and a significantly lower number of telogen-staged hair follicles (p<0.05). Administration of SHED-CM to C3H/HeN mice resulted in a significantly faster stimulation of hair growth in comparison to HFSC-CM (p<0.05), while the duration taken for complete hair coverage was similar for both CM sources. Thus, SHED-CM carries the potential to stimulate hair growth which can be used as a treatment tool for alopecia

Modulator Therapy in Cystic Fibrosis Patients with cis Variants in F508del Complex Allele: A Short-Term Observational Case Series

Previous studies reported the influence of cis variants in F508del cystic fibrosis (CF) patients in their responses to CFTR modulators. The current study is a prospective, observational study involving three patients with CF and pancreatic insufficiency, carrying a complex allele including F508del with A238V, I1027T, or L467F. We report clinical data before and after 4 weeks of treatment with tezacaftor (TEZ)/ivacaftor (IVA), elexacaftor (ELX)/TEZ/IVA, and lumacaftor (LUM)/IVA for patients with complex alleles A238V, I1027T, and L467F, respectively. The 50-year-old patient bearing F508del;A238V/D1152H showed a normal sweat test (13 mEq/L) and improvements in forced expiratory volume in the first second (FEV1) (+7 points), body mass index (BMI) (+0.85), and respiratory CF Questionnaire-Revised (CFQ-R) domain (+22.2 points). The 12-year-old patient bearing F508del;I1027T/R709X showed an improvement in a sweat test (-40 mEq/1), FEV1 (+9 points) and the respiratory CFQ-R domain (+16.7 points). No changes in outcomes were observed for the 6-year-old patient F508del;L467F/F508del. Our data highlight that the reported variants do not modify the phenotypic expression of F508del. Searching L467F is crucial in CF patients with F508del nonresponsive to ELX/TEZ /IVA. Further data are needed to evaluate the clinical effect of these variants after a longer follow up

Rescue of multiple class II CFTR mutations by elexacaftor+ tezacaftor+ivacaftor mediated in part by the dual activities of Elexacaftor as both corrector and potentiator

Positive results in preclinical studies of the triple combination of elexacaftor, tezacaftor and ivacaftor, performed in airway epithelial cell cultures obtained from patients harboring F508del-CFTR, translated to impressive clinical outcomes for subjects carrying this mutation in clinical trials and approval of TRIKAFTATM Encouraged by this correlation, we were prompted to evaluate the effect of the elexacaftor, tezacaftor and ivacaftor triple combination on primary nasal epithelial cultures obtained from individuals with rare Class II cystic fibrosis causing mutations; G85E, M1101K and N1303K for which TRIKAFTATM is not approved. Cultures from individuals homozygous for M1101K responded better than cultures harboring G85E and N1303K after treatment with the triple combination with respect to improvement in regulated channel function and protein processing. A similar genotype specific effect of the triple combination was observed when the different mutations were expressed in HEK-293 cells, supporting the hypothesis that these modulators may act directly on the mutant proteins. Detailed studies in nasal cultures and HEK-293 cells showed that the corrector: elexacaftor, exhibited dual activity as both corrector and potentiator and suggested that the potentiator activity contributes to its pharmacological activity. These preclinical studies using nasal epithelial cultures identified mutation genotypes for which elexacaftor, tezacaftor and ivacaftor may produce clinical responses that are comparable to, or inferior to those observed for F508del-CFTR

The CFTR Mutation c.3453G &gt; C (D1152H) Confers an Anion Selectivity Defect in Primary Airway Tissue that Can Be Rescued by Ivacaftor

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene variant, c.3453G &gt; C (D1152H), is associated with mild Cystic Fibrosis (CF) disease, though there is considerable clinical variability ranging from no detectable symptoms to lung disease with early acquisition of Pseudomonas aeruginosa. The approval extension of ivacaftor, the first CFTR modulator drug approved, to include D1152H was based on a positive drug response of defective CFTR-D1152H chloride channel function when expressed in FRT cells. Functional analyses of primary human nasal epithelial cells (HNE) from an individual homozygous for D1152H now revealed that while CFTR-D1152H demonstrated normal, wild-type level chloride conductance, its bicarbonate-selective conductance was impaired. Treatment with ivacaftor increased this bicarbonate-selective conductance. Extensive genetic, protein and functional analysis of the nasal cells of this D1152H/D1152H patient revealed a 90% reduction of CFTR transcripts due to the homozygous presence of the 5T polymorphism in the poly-T tract forming a complex allele with D1152H. Thus, we confirm previous observation in patient-derived tissue that 10% normal CFTR transcripts confer normal, wild-type level chloride channel activity. Together, this study highlights the benefit of patient-derived tissues to study the functional expression and pharmacological modulation of CF-causing mutations, in order to understand pathogenesis and therapeutic responses